holds various files of this Leiden University
: Vrij, Jeroen de
through Genetic Capsid Modifications
of an oncolytic adenovirus is illustrated by the virus particles attacking the crab. The crab is the international symbol of cancer. Cancer was originally named karkinoma (Greek for krab) by Hippocrates, to whom the growth of a tumor with its sprouting blood vessels reminded on the legs and claws of a crab. As indicated, different types of capsid modifications are being explored to establish tumor-targeting, for example through adding a heterologous polypeptide to the fiber protein or to protein IX. The staircase symbolizes the ‘road towards successful oncolytic virus therapies’. The helical form of the staircase illustrates the importance of introducing genetic modifications in the DNA genome of oncolytic adenoviruses. Copyright © 2012 J. de Vrij, Zoeterwoude, The Netherlands. All rights reserved. No part of this publication may be reproduced or transmitted in any form, without permission from the copyright owner.
The cover includes modified art work from Aruana16 (the crab) and Megainarmy (the spiral stairs) (copyrights were obtained at www.shutterstock.com), and from Jort Vellinga (adenovirus particles) (copyrights were obtained from J. Vellinga).
Layout & printing: Off Page, www.offpage.nl
through Genetic Capsid Modifications
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof. mr. P.F. van der Heijden,
volgens besluit van het College voor Promoties te verdedigen op donderdag 10 mei 2012
klokke 13:45 uur
Jeroen de Vrij
Promotor: Prof. dr. R.C. Hoeben Overige leden: Prof. dr. A.J. van Zonneveld
Prof. dr. C.H. Bangma (Erasmus Medisch Centrum, Rotterdam) Dr. G. van der Pluijm
The research presented in this thesis was performed at the department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
Chapter 1 General introduction 7
Chapter 2 Adenovirus-derived vectors for prostate cancer gene therapy 31
Chapter 3 Efficient incorporation of a functional hyper-stable single-chain
antibody fragment protein-IX fusion in the adenovirus capsid 51
Chapter 4 Adenovirus targeting to HLA-A1/MAGE-A1-positive tumor cells by fusing a single-chain T-cell receptor with minor capsid protein IX 65
Chapter 5 A cathepsin-cleavage site between the adenovirus capsid protein IX and a tumor-targeting ligand improves targeted transduction 87
Chapter 6 An oncolytic adenovirus redirected with a tumor-specific
T-cell receptor 107
Chapter 7 Enhanced transduction of CAR-negative cells by protein IX-gene deleted adenovirus 5 vectors 125
Chapter 8 General discussion 145
Addendum Summary 157
Nederlandstalige samenvatting 159
List of publications 162
tAble Of COntents
1.1 Aims and outline of this thesis 9
1.2 Biology of Human Adenovirus Type 5 10 1.2.1 Virion architecture 11 1.2.2 Cellular infection route 12
1.2.3 Replication 13
1.2.4 Capsid protein IX 14
1.1 AIMs And OutlIne Of thIs thesIs
1. To establish the production of HAdV-5 particles decorated with protein IX-fused polypeptide ligands that have proven potential for tumor targeting, such as single-chain antibody fragments, single-single-chain T-cell receptors, or Affibody molecules.
2. To investigate the targeting efficacy and specificity of protein IX-ligand decorated HAdV-5 vectors to tumor cell lines.
3. To investigate the targeting of HAdV-5 to cancer-testis antigens through fusing a single-chain T-cell receptor with protein IX or fiber molecules.
4. To analyze the effect on transduction of incorporating cathepsin-cleavage sites in between HAdV-5 protein IX and its fused targeting ligand.
5. To obtain insight into the biological consequences of protein IX modification.
Chapter 1 provides a general introduction on HAdV-5, which is the best-studied adenovirus serotype and the serotype most-often used for the construction of oncolytic vectors for cancer gene therapies. Important aspects on the biology of HAdV-5 are summarized, including the virion architecture, the infection route, and the replication mechanism. A separate paragraph is devoted to the minor capsid protein IX of HAdV-5, taking into account the important role of this protein in this thesis. Chapter 1 also provides a general overview on oncolytic adenovirus vectors. Vector modification strategies aiming at improved efficacy are described, as well as strategies for reducing transduction of non-target tissues. The ins and outs are provided for replication-deficient HAdV-5 vectors, as well as for the more recently developed Conditionally Replicating Adenoviruses (CRAds).
In Chapter 2 the most recent advances in oncolytic adenovirus technology are described, focusing on vectors for prostate-cancer treatment. The most prominent bottlenecks for successful cancer gene therapy with oncolytic viruses are reviewed, and potential solutions to overcome these hurdles are outlined.
Chapters 3, 4, and 5 describe the usability of the adenovirus minor capsid protein IX as an anchor for genetically fusing tumor targeting ligands.
The feasibility of fusing large and complex polypeptides to protein IX is described in Chapter 3. As a model ligand the hyper-stable single-chain antibody fragment 13R4 was chosen, which binds with high affinity to β-galactosidase. Incorporation of protein IX-13R4 polypeptides in the virus capsid was achieved with our previously developed “protein-IX screening” system, encompassing the transduction of a protein IX-13R4 producing helper cell line with a protein IX gene-deleted HAdV-5 vector, followed by harvesting and purification of the progeny viruses. Incorporation efficiency and functionality of 13R4 in the capsid of the HAdV-5 vector is discussed.
against the CT antigen MAGE-A1, in complex with HLA-A1. Efficacy of targeting to HLA-A1/MAGE-A1 positive melanoma cell lines is described, as well as various assays to analyze the specificity of targeting.
Chapter 5 describes the results on HAdV-5 viruses targeted to tumor cells through fusion of a high-affinity binding Affibody molecule to protein IX, and the effects of incorporating a cathepsin-cleavage site (ccs) in between protein IX and the Affibody molecule. Previous findings by us and by others suggested that protein IX-mediated targeting using ‘high-affinity binders’ (like Affibody molecules) as ligand is limited by inefficient release of protein IX-fused ligands from their cognate receptors in the endosome. This would result in inefficient endosomal escape of the virus particles. Chapter 5 comprises an extensive comparison between HAdV-5 viruses containing either wild type protein IX, protein IX-Affibody, or protein IX-ccs-Affibody in the capsid. The transduction efficiency is compared in monolayer cultures, 3-dimensional spheroid cultures, and in SKOV-3 tumors grown on the chorioallantoic membrane of embryonated chicken eggs.
In addition to the analyses of the protein IX-scTCR loaded HAdV-5 vectors, as described in Chapter 4, the usability of the HLA-A1/MAGE-A1 specific scTCR for HAdV-5 targeting was also tested in the context of fusion with the fiber protein (Chapter 6). The adenoviral fiber knob, which is responsible for attachment to the Coxsackie virus and Adenovirus Receptor (CAR) on target cells, was replaced by the scTCR molecule and an extrinsic trimerization motif in a replication-competent HAdV-5 vector. The efficacy and specificity of targeting is presented through comparison of cell killing in a panel of melanoma cell lines.
Functional consequences of deleting the protein IX gene from HAdV-5 vectors are described in Chapter 7. The findings provide novel insights into the biological role of protein IX, and may be of relevance for future development and clinical implementation of protein IX-modified HAdV-5 vectors.
Chapter 8 provides a general discussion on the potency of protein IX-mediated tumor targeting for the development of improved oncolytic HAdV-5 vectors. Recommendations for further preclinical studies are included. Also, an overview is given on the newest insights and developments in preclinical testing of oncolytic AdV vectors in general. The anticipated essence of various model systems for future vector analyses is described.
1.2 bIOlOGy Of huMAn AdenOVIrus tyPe 5
can cause considerable morbidity, especially in individuals who are compromised immunologically (e.g. transplant patients) or nutritionally (e.g. gastrointestinal infections in children in the developing world).
Adenoviruses are icosahedral, non-enveloped viruses of approximately 90 nm in size, belonging to the largest non-enveloped viruses. Recently, the structure of a HAdV has been solved at the atomic level, providing the largest high-resolution model ever.1,2 Research on adenoviruses has yielded ample knowledge on various cell biology
mechanisms, such as RNA splicing.3 Also, laboratory experiments on adenoviruses and
their derived vectors has come along with the development of important molecular-biological techniques like the calcium-phosphate DNA transfection method.4
Various aspects, including the relative safety, the well-known biology, and the suitability for genetic modification, have made vectors derived from HAdV type 5 (belonging to the species C HAdVs) the currently most-often used vehicles for viral gene-delivery. HAdV-5 vectors have been used extensively as vaccine, and have shown great potential for ex vivo and in vivo gene therapies for treatment of hereditary diseases or cancer.
1.2.1 Virion architecture
A fully mature HAdV-5 particle, with an approximate mass of 150 MDa, consists of an icosahedral capsid of 20 facets and a core, as schematically depicted in Fig. 1. The pentons, hexons and fibers form the so-called major proteins of the adenovirus capsid. The minor proteins of the capsid are proteins IIIa, VI, VIII and IX. The core consists of a double-stranded linear DNA genome (36 kb in size) and of several proteins: proteins IVa2, V, VII, terminal protein (TP), mu, and the adenovirus protease.5,6 The
HAdV-5 genome contains transcriptional units referred to as early (regions E1 to E4), intermediate (regions pIX and IVa2) and late (regions L1 to L5) depending on their temporal expression, relative to the onset of viral DNA replication.
The fiber protein in the capsid has been identified as the main cell binding protein, with the cell surface protein bound being the coxsackievirus and adenovirus receptor (CAR).7 CAR binds to the fiber knob domain, which is located at the carboxyl-terminus
of the fiber shaft domain. The fiber tail is mounted on the vertex protein penton base. Penton base is located on each vertex and has protruding arginine-glycine-aspartic acid (RGD)-domains that are involved in secondary cell binding and the initiation of virus endocytosis through integrin binding.8,9 Penton base has also been
suggested to play a role in endosomal escape of virus particles, evidenced by the blocking of adenovirus induced endosomal lysis by α-penton antibodies and addition of soluble penton.10,11 The bulk of the capsid consists of 240 copies of trimeric hexon,
contributing to approximately 50% of the mass of the capsid.12 The hexon content of
a capsid can be divided in two subgroups. The groups of nine (GON) are a group of hexons that is frequently observed after dissociation of the capsid.13 The non-GON
belonging hexons are the peripentonal hexons. These are, as the name suggests, the hexons in direct contact with the penton bases. Each trimeric hexon has three towering structures on top, with a cavity in between that can be bound by coagulation factor X in HAdV-5.14,15
for packaging the viral DNA as well as capsid maturation and localizes on the inner side of the pentons.16 Protein VI has been localized to the inner cavity of hexons and has
a function in hexon transport into the nucleus during virus assembly, as well as being involved in the lysis of endosome membranes.17-19 Protein VIII (assigned to the inner
capsid) is thought to provide the capsid stability but the exact functions remains to be solved.20 A description on the smallest but most abundant minor capsid protein, protein
IX, is provided in more detail below, since this protein plays a major role in this thesis.
1.2.2 Cellular infection route
Different receptors are involved in adenovirus cell binding.21 The main receptor
for adenoviruses is CAR, which binds to the fiber knob.7 Alternatively, species B
adenoviruses utilize different receptors, that is, CD46 for species B1 (serotypes 16, 21, 35, 50), desmoglein 2 for species B2 (serotypes 3, 7, 14), and CD46 or desmoglein 2 for species B3 (serotype 11).22 Furthermore, cell surface sialic acid molecules can be utilized
as a receptor by various serotypes, such as HAdV-37 from species D.23,24 Recently, a
remains to be established how important the latter mechanism is for transduction of liver cells in humans. After the initial docking of a HAdV-5 particle to its primary receptor, various secondary interactions can occur, the most prominent one being the interaction of RGD domains of penton base with integrins αvβ3 orαvβ5.8,9 Also
heparan sulphate proteoglycans have been identified as cell surface molecules for secondary interactions, through binding to the fiber shaft.26 Shortly after cell binding
the fiber is shed.27,28 The interaction of penton base RGD domains with cellular
integrins induces a conformational change of penton base monomers, suggested to be necessary for fiber release or for breaking contacts with the surrounding hexons, allowing the fiber or the penton base to be released.29 Integrin aggregation
through penton-base binding localizes the virus to clathrin coated endosomes, the main entry route for adenovirus.30 Integrin binding also activates various intracellular
signaling routes, including the MAPK/p38 and the p85/p110 PI(3) kinase routes, which is thought to influence susceptibility of adenovirus uptake by the canonical routes and by other endocytosis pathways such as macropinocytosis.31,32 Once the virus is
in the clathrin-coated endosome and the environment acidifies, the release of pVI triggers endosomal escape.18,19 Once in the cytosol, HAdV-5 binds molecular motors
that move over microtubules; a minus end directed motor (kinesin) and a plus end directed motor (dynein). These motors are engaged in a tug of war principle, resulting in speeds of movement of micrometers per minute.33,34 Hexon seems to be the protein
responsible for binding kinesins and dyneins.35 Whether the movements of the viral
particle occur according to a stochastic tug of war principle or whether all movements are coordinated is still subject of debate.36 Alternatively, microtubule independent
transport has been reported, suggesting other cytosolic transport mechanisms.37,38
When the partially dismantled HAdV-5 particle reaches the nucleus, it binds the nuclear pore complex and is further dismantled.39 Subsequently, the viral DNA is imported and
transcription of genes can be initiated.
The adenoviral genes can be classified in three groups, based on their time of transcription after infection: the early genes (E1A, E1B, E2, E3, and E4), the intermediate genes (pIX and IVa2), and the late genes (L1 to L5).40 Genes are
1.2.4 Capsid protein IX
The 14.3 kDa protein IX is the smallest of the HAdV capsid proteins. Protein IX is unique to the Mastadenoviruses and is, in contrast to the other capsid proteins, absent in the other adenovirus genera. During the viral replication cycle, the transcription of protein IX messenger RNAs is, for unknown reasons, initiated relatively early after infection, as compared to the mRNAs of the other capsid proteins.40 Each virus facet
contains 12 molecules of protein IX, resulting in a total of 240 molecules per virion. The protein has three conserved regions, as shown by amino acid alignment, located at the amino-terminus, the middle part, and the carboxyl-terminus of protein IX. The amino-terminal regions of protein IX are positioned in the cavities between the hexon tops of hexons that belong to a GON. Each cavity contains the amino-termini of three molecules of protein IX.41 The recent determination of the HAdV-5 structure,
by means of cryoelectron microscopy and x-ray analyses, has revealed the carboxyl-terminus of protein IX to be present in the capsid as quadrimeric coiled-coils, at a location previously assigned to the minor capsid protein IIIa.1,2,42,43 The orientation of
the four coiled-coils is parallel for three of the coils, originating from one facet of the capsid, and anti-parallel for one coil, originating from a neighbouring facet.1,2,43 Taken
together, these assignments result in a large network of protein IX, spanning the entire capsid (Fig. 2).
Despite many years of research the definite function of protein IX is still to be assigned. The protein highly likely acts as capsid cement, since deletion of the protein causes thermal instability.44 The amino-terminus appears to be exclusively responsible
for incorporation of protein IX in the virus capsid and for providing the capsid its thermal stability, probably through stabilizing the GONs.45 Besides its postulated role
in providing the virion stability, protein IX seems to play a role in viral DNA packaging, as suggested by the finding that viruses lacking protein IX have a strong reduction in infectivity if the genome is larger than 95% of wild-type size (>35 kb).46,47 The
carboxyl-terminus of protein IX, which can be deleted from HAdV-5 without affecting thermostability, has been suggested to be essential for post-infection interactions of protein IX with cellular factors, resulting in a stimulatory effect on promoters of early viral genes.48 However, this effect, which was found in a non-viral context, was
subsequently toned down by another study, showing no significant effect of protein IX on viral transcription in the context of viral infection.49
Interestingly, protein IX was reported to form clear amorphic bodies in the nucleus, strongly resembling the so-called PML (promyelocytic leukemia protein) bodies in terms of size and protein contents (including PML and sp100).50 Deleting
the carboxyl-terminus of protein IX abrogated the capacity of protein IX to form PML bodies. Appreciating the importance in cellular biology of PML bodies, for example functioning in regulation of cell cycle and cell growth, it was tempting to speculate on a role of (the carboxyl terminus of) protein IX in host cell modulation.50 However, such
Figure 2. Capsid structure of HAdV-5. (a) Protein density on an exterior region of the capsid, roughly corresponding to one icosahedral facet. The model was created through overlaying a cryo-electron microscopy model of the entire virus particle (at 6-Å resolution) with X-ray crystallography structures of individual proteins. Penton base monomers are indicated in yellow, and hexons are indicated in green (position 1), cyan (position 2), blue (position 3), and magenta (position 4). Protein IX densities, as four trimeric regions and three helical bundles, are indicated in red. Hexons belonging to a group of nine (GON) are marked with an asterisk (*). Figure adapted from Saban et al.42 (b) CryoEM (at 3.6-Å resolution) reveals a physical network of protein IX in the capsid, lashing together hexons into GON tiles. Left insets: Ribbon models of the N-terminal domains of three protein IX monomers (blue, green, and red), overlaying the models of three adjacent hexon monomers (H2, H3, and H4) (gray). The N-terminus of protein IX is in close proximity to the FG2 region of a hexon monomer (lower left inset). Right insets: Ribbon models of the C-terminal domains of protein IX. Four C-terminal domains form a bundle consisting of three parallel α-helices and one antiparallel α-helix. The helices are linked by a ladder of hydrophobic residues (leucines and valines) (magenta). Bottom inset: Ribbon model of protein IX showing three distinguished domains as well as the N-joint region. Figure adapted from Liu et al.1
IX appeared not to co-localize with PML. In the context of HAdV-5 infection in these primary cell cultures, protein IX localizes to the nucleus, regularly forming ring-like structures. In contrast, and in line with the results published by Rosa-Calatrava et al.,50
protein IX formed nuclear bodies in transformed cell lines like A549 alveolar epithelium cells. These observations argue against a role of protein IX in nuclear sequestration of PML in non-transformed cells.
1.3 AdenOVIrus VeCtOrs fOr CAnCer therAPy
Genetically modified adenoviruses have been explored extensively as gene-transfer vehicle for the purpose of gene therapy or vaccination.53 Several characteristics
make adenoviruses highly suitable as gene-transfer vehicle: a relatively low level of pathogenicity; a high stability of the viral DNA genome, thereby preventing the
Figure 3. Subcellular localization of protein IX in mesenchymal stem cells, as visualized by immunohistochemistry. (a) Detection of protein IX and promyeolocytic leukemia (PML) protein after establishing lentiviral vector-mediated heterologous expression of protein IX. The cells were fixed with acetone-methanol (1:1). Staining was performed by means of primary incubation with the antibodies
development of heterogeneous populations of ‘quasispecies’; the ability to transduce dividing as well as quiescent cells; the well known biology and uncomplicated genetic modification; the availability of technologies for production of clinical-grade virus batches with high titers and high purity. Adenoviruses can be used either as replication-deficient or replication-competent vectors. The replication-replication-deficient vectors can be used as gene delivery vehicle for gene augmentation therapy (e.g. through delivery of genes that are mutated in the vector-receiving patient) or cancer therapy (e.g. through delivery of prodrug-activating genes). Also, replication-deficient vectors are being used (with proven efficacy and safety) as vaccine vector to induce immune responses against antigenic polypeptides that are displayed on the viral capsid or encoded for by the viral vector.54 More recently, replication-competent adenovirus vectors, or
Conditionally Replicating Adenoviruses (CRAds), have been developed, exploiting the lytic infection cycle of the virus to kill tumor cells. Various modifications can be introduced to CRAds to provide tumor cell selective replication.
Currently, the large majority of adenovirus vectors for cancer therapies are derived from HAdV of serotype 5, mainly as a result of its well known biology and its proven safety. In the next paragraphs a general overview is given on the development of HAdV-5-derived vectors for cancer therapy, describing replication-deficient as well as replication-competent vectors. An extensive outline is provided on rational design approaches towards improved oncolytic HAdVs, such as capsid modifications for targeting and detargeting purpose. Preclinical developments on random approaches, such as bioselection with mutagen-induced viral libraries, are summarized as well.
1.3.1 Replication-deficient vectors
Different types, or ‘generations’, of replication-deficient HAdV-5 vectors have been developed. The first-generation vectors have the E1 region deleted.55 This deletion
renders the recombinant virus replication-defective, providing an important safety feature. The production of E1-deleted vectors is dependent on specialized helper cells that provide the E1 functions in trans. The most frequently used helper cell lines are the 293 cell line (human embryonic kidney cells transformed with sheared HAdV5 DNA)56 and the 911 and PER.C6 cell lines (human embryonic retinoblasts transformed
with a plasmid carrying a defined portion of the adenovirus genome).57,58 By combining
removal of the E1 region with removal of the E3 region, which encodes for proteins involved in evasion of the immune system and is dispensable for vector growth in vitro, approximately 7500 base pairs of heterologous DNA can be accommodated in HAdV-5 vectors.
The first-generation HAdV-5 vectors appeared to be suboptimal for certain gene-delivery applications, mainly as a result of the induction of a strong cell-mediated immune response.59 These responses appeared to be a result of viral protein expression.
As a consequence second-generation HAdV-5 vectors were made in which deletions or mutations were introduced in the E2 and E4 region. These modifications resulted in a substantial reduction of the cellular immune response and, as a consequence, prolonged transgene expression.55 Also third-generation or ‘high-capacity’ HAdV-5
vectors have been developed, which are devoid of all viral genes and can accommodate up to 35 kilo bases of heterologous DNA.55,60 The only remaining viral sequences are
high-capacity vectors requires the usage of a helper virus to provide all viral functions and structural proteins in trans. Elegant systems have been developed to prevent the presence of helper virus contaminants in the final high-capacity vector preparation. As intended, high-capacity HAdV vectors have a strongly improved duration of transgene expression as compared to first- or second-generation HAdV vectors, as a result of a reduced cellular immune response.55
As evidenced by various studies, the first-generation HAdV-5 vectors are inferior to the higher-generation vectors in these gene-delivery applications that require the in vivo expression of large heterologous genes for prolonged times. However, the usage of replication-deficient vectors for the (short-term) expression of oncolytic genes in cancer cells not necessarily requires the usage of higher-generation vectors. First-generation vectors can perfectly accommodate the majority of anti-tumor transgenes and, importantly, induce immune responses that might be of benefit for the efficacy of the therapy (e.g. through induction of a cellular immune response against tumor antigens). Examples of anti-tumor transgenes are genes encoding for prodrug converting enzymes (e.g. the Herpes Simplex Virus thymidine-kinase (HSV-TK) for activating gancyclovir, the bacterial nitroreductase for activating CB1954), immune stimulatory cytokines (IL-2, IL-12, GM-CSF, IL-24), or apoptotic proteins (e.g. p53).61
Specificity of transgene expression can be provided through the inclusion of tissue-specific promoters.
To improve the specificity and efficacy of AdV vectors, a large variety of capsid modifications is being pursued. Such modifications aim, on one hand, on the enhancement of tumor cell transduction (e.g. through fusing tumor-targeting polypeptides to the viral capsid) and, on the other hand, on reduced transduction of non-target cells or tissues. Transductional targeting and detargeting approaches will be discussed in Paragraph 1.2.3.
1.3.2 Conditionally-Replicating Adenoviruses (CRAds)
Past clinical trials have defined major limitations of replication-deficient vectors for cancer gene therapy, as a result of their inability to infect the majority of cells within a clinically presented three-dimensional solid tumor mass.62 Conditionally Replicating
Adenoviruses (CRAds) are designed to overcome this limitation by making use of the natural ability of HAdV-5 to kill their host cells upon its spread throughout a tissue. To provide specificity of cell killing, CRAds are designed in such a way to restrict their replicative ability to tumor cells. Besides the direct lytic effect of CRAds, their induction of cell death might cause anti-tumor immune responses as a positive bystander effect.63,64 Future research is necessary to fully delineate the effects of CRAd
therapies on the patient’s immune system.
CRAds are rendered tumor-specific by taking advantage of cancer-specific cellular changes. It was found by Berk et al. that disruption of the HAdV-5 E1B-55K gene in the mutant dl1520 yielded specific replication in cancer cells.65 Based on previous
experiments showing an ability of E1B-55K to interact with the tumor-suppressor protein p53, it was initially thought that the tumor selectivity of dl1520 was due to p53 being mutated in cancer cells. However, dl1520 was found to replicate in p53-expressing cancer cells as well.66,67 Despite the controversy on the mechanism behind the tumor
vectors. The dl1520 virus has been tested in a variety of cancer clinical trials (under its commercial name ONYX-015) including head and neck cancer,68 oral carcinoma,69
colorectal carcinoma metastases to the liver,70 hepatocellular carcinoma71 and glioma.72
These studies have demonstrated safety, with well tolerated doses of up to 2 x 1012
particles (by various routes of injection) and tumor-selective replication. The efficacy as a single agent has been relatively limited to date (0-14% local tumor regression rates), but encouraging anti-tumor activity has been demonstrated in combination with chemotherapy.73 A very similar virus, H101, has been registered for clinical use in China.
Cancer cell-specific CRAds can also be made by mutating the E1A gene.74 A 24-bp
deletion was found to prevent E1A from binding to the cellular protein Rb for induction of the cellular S-phase. As a consequence, replication depends on the inactivation of Rb through other means (e.g. hyperphosphorylation) which is the case in most types of cancer. Clinical safety and efficacy of E1AD24 CRAds is subject of current research. Recently, the maximum tolerated dose, toxicity spectrum, clinical activity, and biological effects were evaluated for a E1AD24 CRAd (named Ad5-Δ24-RGD) in patients with ovarian cancer.75 Besides having the 24-bp deletion, to establish
tumor-selectivity, this CRAd also contained an RGD-domain fused to the fiber, to enhance efficacy of the treatment. The approach appeared to be safe, and a minor antitumor response was found.
As an alternative to the E1 deletions, CRAds can be created through the incorporation of tissue-specific promoters to control the expression of essential viral genes. As an example, expression of the viral E1A gene can be controlled by the recombinant prostate-specific PPT sequence, which is composed of a prostate-specific antigen (PSA) enhancer, a prostate-specific membrane antigen (PSMA) enhancer and a T cell receptor gamma-chain alternate reading frame protein (TARP) promoter.76
As a result, the AdV vector replicates exclusively in normal and neoplastic prostate epithelial cells.
A large variety of modifications is being explored, to further improve the efficacy and specificity of CRAds. Similar to the replication-deficient vectors, the CRAd genome can be armed with ‘cell killing transgenes’, which may improve the efficacy of tumor eradication. As an example, arming the dl1520 virus with the HSV-TK/ Ganciclovir system results in increased survival rates in mice with subcutaneous colon cancer xenografts.77 Alternatively, CRAds with improved clinical performance may be
obtained by the insertion of genes coding for proteins with antitumor effect on the tumor micro-environment, such as angiogenesis inhibition or immune activation.78 To
improve the specificity and safety of CRAds, their replication can be blocked in non-target cells by incorporating microRNA (miRNA)-binding sequences in viral genes. In this way, multiple binding sites for a hepatocyte-specific miRNA, mir-122, have been placed in the 3’ untranslated region of the E1A gene of a CRAd, leading to the absence of E1A gene expression (and viral replication) in murine hepatocytes and a significant reduction in hepatotoxicity.79,80
1.3.3 Capsid modifications for targeting and detargeting
Studies on oncolytic HAdV-5-based vectors, in preclinical- as well as early phase clinical settings, have demonstrated the necessity of introducing modifications in the viral capsid to improve the efficacy and safety in the complex environment of a patient’s tumor.
One important efficacy-limiting aspect is the low-level expression of the CAR receptor on many tumor cells, necessitating capsid modifications to alter the wild-type tropism of HAdV-5.81,82 Development of modified vectors that can infect
CAR-negative cells has mainly focused on the genetic incorporation of heterologous ligands in the fiber protein, or on ‘fiber-swap’ strategies in which the HAdV-5 fiber is replaced by a fiber from another HAdV serotype.83 Although effective, the applicability
of incorporating large and complex ligands (e.g. single-chain antibody fragments) into fiber locales might be limited, since such modifications in many cases result in virus replication with low titers.21 This drawback has prompted the identification of other
capsid proteins (hexon, penton base, protein IIIa, and minor capsid protein IX) as usable locales for incorporating heterologous peptides (reviewed by Vellinga et al.5).
An interesting locale for the fusion of polypeptides is the minor capsid protein IX. Fusing polypeptides to the carboxyl-terminus of protein IX does not reduce the viral titers upon in vitro production, and has no effect on the stability of the virus particles.45 It was found that the presentation of protein IX-fused peptides can be
improved through incorporating a 75-Ångstrom alpha-helical spacer in between protein IX and the peptide.84 Using the protein IX-spacer sequence as anchor, highly
efficient coverage of the virions with heterologous peptides can be obtained with incorporation efficiencies close to the theoretical maximum of 240 molecules per virion, depending on the size and complexity of the polypeptide. The feasibility of targeting HAdV-5 to tumor cells through fusing tumor-targeting ligands to protein IX has subsequently been investigated, as described in detail in this thesis.
Nowadays, elegant systems are available for creating genetically modified HAdV-5 vectors, which enable cloning and recombination steps in a bacterial context instead of in human cell lines. Still, genetic modification of the HAdV-5 genome is a time- and effort consuming process, limiting the rapid screening of polypeptide moieties for their capsid incorporation ability. For this reason, screening-facilitating systems have been developed based on the propagation of HAdV-5 vectors on cell lines expressing heterologous peptides fused to a capsid protein. Such systems have been used successfully for expedited functional assessment of modified variants of protein IX and fiber.85,86
An alternative to genetically modifying the viral vector for transductional purposes has been provided by ‘adapter strategies’, applying bispecific targeting moieties that on one hand bind to the virus (in general to the fiber knob domain), and on the other hand to a molecule on the target cell. As an example, HAdV-5 vectors have been efficiently retargeted to HER2/neu expressing tumor cells through using designed ankyrin repeat proteins (DARPins) as bivalent adapter molecules.87 The
ligands. However, special care will be required to ensure the preparation of clinical batches with defined characteristics, for example assuring low variability of ligand incorporation efficiencies between different vector preparations.
The above described modifications aim at enhanced transduction of tumor cells through coupling tumor-targeting ligands to the HAdV-5 capsid. Another strategy to improve the potency of oncolytic HAdV-5 vectors is by detargeting the vector from non-target tissues. As discussed in more detail in the next chapter, last years have witnessed an enhanced understanding on the in vivo mechanisms behind the disappointing anti-tumor efficacies of oncolytic HAdV-5 vectors in early-phase clinical trials. One aspect thwarting effective therapy is the high prevalence of pre-existing humoral immunity against HAdV-5 in the human population, resulting in rapid clearance of the vectors from the blood. Additionally, strong innate immune responses, e.g. by natural killer cells, are observed after intratumoral or systemic injection of oncolytic HAdV-5 vectors. Another bottleneck, especially hampering the efficacy of systemically delivered oncolytic HAdV-5 vectors, is the rapid clearance from the blood stream as a result of sequestration in the liver. It has recently been found that binding of HAdV-5 to blood coagulation factor (F) X, results in uptake of the vectors by hepatocytes in the liver.14,15
FX appears to bind to viral capsid epitopes of the hexon protein, and bridges the virus to heparan sulphate proteoglycans on hepatocytes. Besides the FX-hepatocyte mediated removal of oncolytic HAdV-5 vectors, the vectors are also cleared form the blood by liver-residing macrophages (Kupffer cells).88 Binding of the vectors to
complement proteins, natural antibodies and platelets seems to play an important role in the uptake by these scavenging macrophages.89,90 Another problem to tackle is
the binding of HAdV-5 vectors to erythrocytes.91,92 This binding appears to be specific
for human erythrocytes, and has therefore not been noticed previously during vector analyses in rodent models. Last years have seen enormous pre-clinical improvements in the efficacy of HAdV-5 based oncolytic vectors, through applying novel types of modifications leading to improved transductional targeting and detargeting. One highly promising example is the ability to genetically modify viral hexon sequences to abolish uptake of virus particles by hepatocytes, thereby enhancing gene transfer to target cells.93,94
Additionally, strategies have been developed to reduce off-target binding by shielding the adenovirus vector particles with chemical polymers.95 In animal models,
this technology significantly increases the circulation time of HAdV-5 vectors in the blood stream, and simultaneously reduces liver toxicity.96,97 Similar to targeting of
the ‘naked’ vector particles, the polymer coatings can also be modified to achieve targeting to tumor cells.98 Research is ongoing to further improve the polymer coating
technology, for example aiming at ‘low pH triggered de-shielding’ to facilitate proper intracellular routing of polymer coated virus particles after their uptake in the endosome. As an alternative to the polymer coatings, ‘carrier cells’ might be utilized to shield HAdV-5 vectors from efficacy-limiting moieties. Various cell types with intrinsic tumor-homing properties, such as mesenchymal stem cells, T cells, and monocytes, are currently under investigation.99
as well as prominent aspects that require further optimization, are outlined in more detail in Chapter 2.
1.3.4 Random approaches to vector development
As described in the previous sections, a plethora of rational design approaches is being pursued to develop AdV vectors with improved performance. However, last years have witnessed a renewed interest in the more traditional ‘directed evolution’ method of oncolytic vector development; the random creation of virus mutants followed by bioselection of the best-performing viruses. This approach has yielded improved oncolytic HAdVs with genomic mutations that would have never been picked up using rational design approaches. These findings not only benefit to the development of improved oncolytic HAdVs, but also enhance our knowledge on HAdV biology.
Following a mutagenesis and bioselection approach, Yan et al. plaque purified two mutants, ONYX-201 and ONYX-203, from a pool of randomly mutated HAdV-5 that was repeatedly passaged in the human colorectal cancer cell line HT29.100 The
mutants replicated more rapidly in HT29 cells than wild-type HAdV-5, and lysed HT29 cells up to 1,000-fold more efficiently. The enhanced cytotoxicity was also observed in other human cancer cell lines, but not in a number of normal primary human cells, indicating a strong enhancement of the therapeutic index of 201 and ONYX-203. Although the virus mutants contained multiple single-base-pair mutations, they shared a mutation at nucleotide 8350, which was shown to be essential for the observed phenotype. This mutation was mapped to the i-leader sequence of the HAdV-5 genome, which is (for unknown reasons) present as a 440-nucleotide leader sequence in the majority of HAdV-5 major-late transcripts. The i-leader contains an open reading frame encoding for a 16 kDa-sized protein.101 The mutation at nucleotide
8350 introduces a stopcodon, resulting in a truncation of 21 amino acids from the C terminus of the i-leader protein. In parallel to these results, another i-leader mutant HAdV-5 was isolated by Subramanian and coworkers in a screen for large plaques on A549 alveolar epithelium cells.102 Although the exact mechanism behind the improved
oncolytic performance of i-leader mutated HAdV-5 remains to be investigated, the potential of this type of mutation was recently underscored by Van den Hengel et al., who demonstrated enhanced cytopathic activity of i-leader mutated HAdV-5 in glioma cell lines and primary glioma cultures.103
Using similar mutagenesis and bioselection approaches, another type of HAdV-5 mutant with enhanced antitumor efficacy was found by Gros et al.104 The propagation
of a mutagenized HAdV-5 stock in human tumor xenografts led to the isolation of a mutant virus displaying a large-plaque phenotype in vitro and an enhanced antitumor activity in vivo. A truncating mutation in the viral E3-19K gene, resulting in relocalization of the E3-19K protein from the endoplasmatic reticulum to the plasma membrane, appeared to be responsible for the mutant’s enhanced antitumor efficacy. The aberrant protein localization appeared to enhance the cellular influx of calcium ions, thereby deregulating calcium homeostasis and inducing membrane permeabilization.
Recently, Uil et al. presented another type of directed evolution, through serial passaging of HAdV-5 in cancer cells in the context of a ‘sloppy’ viral polymerase protein.105
having a mutation in the single-strand DNA binding region of the exonuclease domain, were exploited to generate HAdV-5 mutants with improved cytolytic activity in tumor cells. A common mutation was identified, located in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, high and untimely expression of ADP was observed, presumably causing the enhanced cytotoxicity.
Kuhn and coworkers have used a directed evolution approach to obtain chimeric oncolytic adenoviruses, that consist of components from different HAdV serotypes.106
An array of serotypes, representing HAdV species B to F, was pooled and passaged on tumor cell lines under conditions that invite recombination. By using this methodology, a highly potent oncolytic HAdV-3/HAdV-11p chimeric virus (named ColoAd1) was obtained. ColoAd1 demonstrated greatly enhanced potency and selectivity, as compared to its parent serotypes and ONYX/015, in colon cancer cell cultures and in a mouse tumor model.
The proven potential of random selection approaches has triggered researchers to combine this type of approach with rational design. As such, improved HAdV vectors targeted to prostate cancer cells have been isolated after genetically incorporating random peptides at viral capsid locales flanking the tumor-targeting polypeptide sequence.107
Directed evolution approaches are expected to lead to the isolation of novel and improved oncolytic HAdVs. Performing such strategies in clinically relevant model systems will be of great interest, acknowledging the large repertoire of efficacy-limiting in vivo aspects of oncolytic viral therapy, with many aspects having non-resolved mechanisms of action.
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J de Vrij1
, ra Willemsen2
, L Lindholm3
, rC hoeben1
and the GIaNt consortium*
1Department of Molecular Cell Biology, Leiden University Medical Center,
Leiden, the Netherlands, 2tumor Immunology Group, Department of Medical
Oncology, erasmus MC-Daniel den hoed, rotterdam, the Netherlands and
3Got-a-Gene aB, Kullavik, Sweden.
human Gene therapy 2010; 21:795-805
ADENOVIRUS-DERIVED VECTORS FOR PROSTATE
CANCER GENE THERAPY
* The GIANT consortium consists of: CH Bangma1, C Barber14, JP Behr11, S Briggs2, R Carlisle3, WS Cheng2, IJC Dautzenberg4, C de Ridder1, H Dzojic2, P Erbacher12, M Essand2, K Fisher13, A Frazier9, LJ Georgopoulos14, I Jennings14, S Kochanek5, D Koppers-Lalic4, R Kraaij1, F Kreppel5, M Magnusson7, N Maitland9, P Neuberg13, R Nugent14, M Ogris10, JS Remy11, M Scaife14, E Schenk-Braat1, E Schooten1, L Seymour2, M Slade14, P Szyjanowicz14, T Totterman2, TG Uil4, K Ulbrich6,
L van der Weel1, W van Weerden8, E Wagner10, G Zuber11.